The displacement mode of chromatography was first recognized in 1906 by Tswett, who noted that sample displacement occurred under conditions of overloaded elution chromatography. In 1943, Tiselius developed the classifications of frontal chromatography, elution chromatography, and displacement chromatography. Since that time most developments and applications, particularly those in analytical chromatography, have taken place in the area of elution chromatography, and indeed the term chromatography without further qualification usually refers to elution chromatography. Nonetheless, while the theory and practice of elution chromatography has dominated the literature for the past fifty years, the theory and practice of displacement chromatography has occupied a small niche in chromatographic science.
The two types of chromatography, elution and displacement, are readily distinguished both in theory and in practice. In elution chromatography, a solution of the sample to be purified (in the case of the present invention, a protein) is applied to a stationary phase, commonly in a column. The mobile phase is chosen such that the sample is neither irreversibly adsorbed nor totally unadsorbed, but rather binds reversibly. As the mobile phase is flowed over the stationary phase, an equilibrium is established between the mobile phase and the stationary phase whereby, depending upon the affinity for the stationary phase, the sample passes along the column at a speed which reflects its affinity relative to other components that may occur in the original sample. The differential migration process is outlined schematically FIG. 1, and a typical chromatogram is shown in FIG. 2. Of particular note in standard elution chromatography is the fact that the eluting solvent front, or zero column volume in isocratic elution, always precedes the sample off the column.
A modification and extension of isocratic elution chromatography is found in step gradient chromatography wherein a series of eluents of varying composition are passed over the stationary phase. In ion exchange chromatography, step changes in the mobile phase salt concentration and/or pH are employed to elute or desorb the proteins.
Displacement chromatography is fundamentally different from desorption chromatography (e.g., affinity chromatography, step gradient chromatography). The displacer, having an affinity higher than any of the feed components, competes effectively for the adsorption sites on the stationary phase. An important distinction between displacement and desorption is that the displacer front always remains behind the adjacent feed zones in the displacement train, while desorbents (e.g., salt, organic modifiers) move through the feed zones. The implications of this distinction are quite significant in that displacement chromatography can potentially concentrate and purify components from mixtures having low separation factors while in the case of desorption chromatography, relatively large separation factors are generally required to give satisfactory resolution.
In displacement chromatography the eluent, (i.e. the displacer) has a higher affinity for the stationary phase than does any of the components in the mixture to be separated. This is in contrast to elution chromatography, where the eluent usually has a lower affinity. The key operational feature which distinguishes displacement chromatography from elution or desorption chromatography is the use of a displacer molecule. In displacement chromatography, the column is first equilibrated with a carrier solvent under conditions in which the components to be separated all have a relatively high affinity for the stationary phase. A large volume of dilute feed mixture can be loaded onto the column and individual components will adsorb to the stationary phase. That is, they distribute from the feed solution onto the stationary phase, and remain there. If all the components are to be resolved by displacement, the carrier solvent emerges from the column containing no sample. The sample now resides on the stationary phase and the position of each component on the column is correlated with its relative affinity for the stationary phase. Conceptually, one can imagine each molecule of the component with the highest affinity for the stationary phase displacing a molecule of a component having a lower affinity at a site on the stationary phase so that the individual components will ultimately be arranged on the column in sequence from highest to lowest affinity.
It will sometimes be advantageous to allow some of the components to pass through the column with the carrier solvent; in this case only the retained feed components will be resolved by displacement chromatography.
Once the sample is loaded on the column, a displacer solution is introduced. The displacer solution comprises a displacer in a suitable solvent. The displacer is selected such that it has a higher affinity for the stationary phase than does any of the feed components. Assuming that the displacer and mobile phase are appropriately chosen, the product components exit the column as adjacent squarewave zones of highly concentrated pure material in the order of increasing affinity of absorption. This is shown schematically in FIG. 3. Following the zones of purified components, the displacer emerges from the column. A typical chromatogram from a displacement chromatography is shown in FIG. 4. It is readily distinguished from the chromatogram of elution chromatography shown in FIG. 2 by virtue of the fact that the displacer follows the sample and that the feed components exit the column as adjacent "square wave" zones of highly concentrated pure material. Finally, after the breakthrough of the displacer, the column is regenerated by desorbing the displacer from the stationary phase to allow the next cycle of operation.
Displacement chromatography has some particularly advantageous characteristics for process scale chromatography of biological macromolecules such as proteins. First, and probably most significantly, displacement chromatography can achieve product separation and concentration in a single step. By comparison, isocratic elution chromatography results in product dilution during separation. Second, since the displacement process operates in the nonlinear region of the equilibrium isotherm, high column loadings are possible. This allows much better column utilization than elution chromatography. Third, column development per se requires less solvent than a comparable elution process. Fourth, displacement chromatography can concentrate and purify components from mixtures having low separation factors, while relatively large separation factors are required for satisfactory resolution in desorption chromatography.
With all of these advantages, one might presume that displacement chromatography would be widely utilized. However, displacement chromatography, as it is traditionally known, has a number of drawbacks vis-a-vis elution chromatography for the purification of proteins The term "protein", as commonly understood in the art and as used herein, refers to polypeptides of 10 kDa molecular weight or more; according to this convention, polypeptides of molecular weight below 10 kDa are commonly referred to as oligopeptides. Two of the major problems are (1) regeneration of the column and (2) the presence of displacer in some of the purified fractions.
Since the displacement process uses a high affinity compound as the displacer, the time for regeneration and re-equilibration can be long compared to elution chromatography. Furthermore, relatively large amounts of solvent are often required during regeneration, effectively reducing any advantage over elution chromatography in solvent consumption.
The second problem, that of contamination by the displacer, has arisen because a common characteristic of displacers used in protein separations has been their relatively high molecular weight. Heretofore the art has taught the use of high molecular weight polyelectrolytes to displace proteins on the assumption that (as explained below) it is necessary to have a large polyelectrolyte in order to ensure a higher binding coefficient than the protein that is to be displaced. High molecular weight displacers exhibit both of the disadvantages enumerated above: they bind tightly to the stationary phase and therefore require heroic conditions for regenerating the column, and traces of the displacer that may contaminate the product fraction are difficult to remove.
Therefore, it would be advantageous to have a class of displacers that did not require extensive regeneration of the column and that could be readily removed from the product protein. There is one example in the art known to applicants of an attempt to use 2 kilodalton poly(vinylsulfonic acid) on polyethyleneimine-coated weak anion exchange resin for the separation of conalbumin from ovalbumin. The experiment appears to have been successful in that the two proteins were separated [See Jen and Pinto Journal of Chromatography 519, 87-98 (1990)]. However the separation appeared to have been effected by a mixed mechanism of elution and displacement chromatography, as discussed in a subsequent paper [see Jen and Pinto Journal of Chromatographic Science 29, 478-484 (1991)] in which the authors abandoned the poly(vinyl sulfate) displacers in favor of higher molecular weight dextran sulfate. In this second paper, Jen and Pinto demonstrate the superiority of the larger dextran sulfate over the smaller polyvinyl sulfate.
In a subsequent article, Jen and Pinto [Reactive Polymers 19, 145-161 (1993), p.147] provide a table of all displacers used for the displacement chromatography of proteins on ion exchange stationary phases. In their discussion of the results, they conclude, as before, that the 2 kDa polyvinyl sulfate partially displaces the second protein and elutes the first.
It has now been surprisingly found that several classes of charged species of very low molecular weight can function very efficiently as displacers for proteins in displacement chromatography.